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Talmadge, 2000; Wehling et al., 2000). Examples of IMA are ... towards a slow muscle phenotype (Talmadge, 2000). ...... Eugene: University of Oregon. Press.
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The Journal of Experimental Biology 208, 3675-3687 Published by The Company of Biologists 2005 doi:10.1242/jeb.01826

Effects of decreased muscle activity on developing axial musculature in nicb107 mutant zebrafish (Danio rerio) T. van der Meulen*, H. Schipper, J. L. van Leeuwen and S. Kranenbarg Experimental Zoology Group, Wageningen Institute of Animal Sciences, Wageningen University, Marijkeweg 40, NL-6709 PG Wageningen, The Netherlands *Author for correspondence (e-mail: [email protected])

Accepted 8 August 2005 Summary The present paper discusses the effects of decreased myofibril arrangement show a less regular pattern. muscle activity (DMA) on embryonic development in the Finally, expression levels of several genes were changed. zebrafish. Wild-type zebrafish embryos become mobile Together, these changes in expression indicate that muscle around 18·h post-fertilisation, long before the axial growth is not impeded and energy metabolism is not musculature is fully differentiated. As a model for DMA, changed by the decrease in muscle activity but that the the nicb107 mutant was used. In nicb107 mutant embryos, composition of muscle is altered. In addition, skin stiffness is affected. In conclusion, the lack of muscle fibre activity muscle fibres are mechanically intact and able to contract, did not prevent the basal muscle components developing but neuronal signalling is defective and the fibres are not but influenced further organisation and differentiation of activated, rendering the embryos immobile. Despite the these components. immobility, distinguished slow and fast muscle fibres developed at the correct location in the axial muscles, helical muscle fibre arrangements were detected and Supplementary material available online at http://jeb.biologists.org/cgi/content/full/208/19/3675/DC1 sarcomere architecture was generated. However, in nicb107 mutant embryos the notochord is flatter and the crosssectional body shape more rounded, also affecting muscle Key words: decreased activity, development, zebrafish, nicb107 fibre orientation. The stacking of sarcomeres and mutant, muscle.

Introduction Embryonic development is not just a sequence of one gene expression following another. Mechanical loading influences development (Bagatto et al., 2001; Cho, 2004; Hutson et al., 2003; Prendergast, 2002; Vandenburgh et al., 1991). Muscle and bone are tissues that generate and support mechanical loads, which also regulate their growth, maintenance and differentiation (Hoppeler and Fluck, 2002; Huiskes et al., 2000; Pette, 2001). We will consider the effects of an absence of mechanical load by immobility on muscle development in a model system for vertebrate development, the zebrafish (Danio rerio Hamilton). Increasing muscle activity by forced swimming in fish stimulates red muscle development, enhances muscle enzyme activity, increases blood oxygen carrying capacity, increases mitochondrial density, improves swimming efficiency and increases hypoxia tolerance (Bagatto et al., 2001; Davison, 1989, 1997; De Graaf et al., 1990; Kieffer, 2000; Pelster et al., 2003). Decreased muscle activity transiently downregulates the activity of the muscle enzyme succinate dehydrogenase (De Graaf et al., 1990). Decreased activity by immobility in zebrafish and Xenopus laevis embryos correlated with

abnormal muscle fibre distribution and axial musculature architecture in some (Droin and Beauchemin, 1974; Granato et al., 1996; Van Raamsdonk et al., 1977, 1982) but not all cases (Granato et al., 1996). The effects of altered muscle activity on gene expression levels during development have been only rarely investigated. The bulk of zebrafish axial muscle consists of fast white fibres. Slow red fibres form a superficial layer, and intermediate pink fibres are located in between (Waterman, 1969). The white trunk axial musculature is arranged in a series of myomeres, which are separated by collagenous sheets, the myosepta. Muscle fibres in adult fish run between the myosepta in a pseudo-helical pattern (Alexander, 1969; Ellerby and Altringham, 2001; Gemballa and Vogel, 2002; Johnston et al., 1995; Mos and Van Der Stelt, 1982; Van der Stelt, 1968; Van Leeuwen, 1999). This pseudo-helical arrangement is thought to be an optimisation for muscle work output (Alexander, 1969) and, as such, may be influenced by mechanical loading. At 18·hours post-fertilisation (18·hpf), zebrafish embryos start making their first feeble movements (Westerfield, 1995). At this age, slow muscle fibres in the

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3676 T. van der Meulen and others zebrafish embryo are adjacent and parallel to the notochord, can be stained for heavy myosin chains and are about to migrate laterally through the somite (Devoto et al., 1996). Fast fibres, which already form the bulk of the muscle mass, do not stain for heavy myosin chains until after 23·hpf (Devoto et al., 1996). Helical arrangements of zebrafish muscle fibres are first observed at 4·days after hatching (Van Raamsdonk et al., 1974). This timeline of development and use of the axial musculature also suggest that early use of the axial musculature is crucial for its proper development (Van Raamsdonk et al., 1977). As the use of the musculature appears crucial for proper development of the axial musculature, lack of use, i.e. immobility, is expected to hamper proper development. We used the nicb107 mutant to study the effect of immobility on axial muscle development. In this mutant, the ␣-subunit of the acetylcholine receptor is defective, which blocks assembly of functional acetylcholine receptors on the muscle fibres (Sepich et al., 1998). As a result of this lack of innervation, the muscle fibres fail to contract in vivo. They are mechanically intact, however, as they are able to contract upon electrical stimulation (Westerfield et al., 1990). In the present paper, we report several effects of immobility on the morphogenetic development of axial musculature in early zebrafish larvae by studying the expression levels of a selection of structural as well as regulatory muscle genes and studying muscle structure at different organisational levels. Materials and methods Animals Embryos from the nicb107 strain were bought from the Zebrafish International Resource Center (ZIRC) in Oregon, USA (NIH-NCRR grant #RR12546) and raised in our facility. Zebrafish embryos were generated by natural spawnings of heterozygous parents. Homozygous mutants were selected on the basis of immobility and an inability to respond to touch. Embryos were reared at 28.5°C and removed from the egg capsule at 24·hpf using needles. Collecting and processing morphometric data Embryos were positioned in sedative (1·g·l–1 MS-222 and 1.5·g·l–1 Na2CO3.H2O) on a 1% agarose gel. Lateral photographs were taken using an Olympus DP50 digital camera mounted on a Zeiss Stemi SV11 microscope with AnalySIS software V3.1 (Soft Imaging System GmbH, Münster, Germany). At 24, 48, 72, 96 and 120·hpf, total body length and, at anus level, muscle height, notochord height, somite size in anterior–posterior direction and angle of the somite were measured from photos using AnalySIS software in both nicb107 embryos and their wild-type siblings. Five to eight animals were used per group per measurement. Statistical differences between wild-type and nicb107 data were detected using the Mann–Whitney U test and were considered significant when P